Autism spectrum disorders (ASDs), or pervasive developmental disorders (PDDs), are a category of complex developmental brain disorders that appear in early childhood, usually before the age of three. ASDs cause difficulties in social, communication and behavioural skills, but affected individuals can manifest a wide range of symptoms, from very mild to severe - hence the term "spectrum".

One in 110 children in the US has an ASD, with four times as many boys than girls being affected. In the last decade, diagnoses have increased tenfold.

Copy number variants (CNVs) – a form of structural variation - are deletions or duplications of genomic segments ranging from several thousand to a few million base pairs. A number of population-based studies have demonstrated that CNVs can affect as much as 12% of the human genome. Large scale studies have gone on to show the importance of CNVs in determining human phenotypic variation and disease susceptibility.

There is strong evidence that rare CNVs play a role in susceptibility to ASDs. A large study published in Nature by the Autism Genome Project (AGP), an international consortium of scientists from more than 60 institutions in 12 countries, compared the DNA of almost 1000 children with ASDs and healthy children, using 1 million single-nucleotide polymorphism (SNP) arrays. The study showed that rare CNVs, possibly acting in tandem, play a significant role in the genetic aetiology of this condition. The rare variants were identified in less than 1% of the normal population but strikingly, occurred almost 20% more frequently in ASD children.

Further recent research has focused on how CNVs mediate a phenotypic effect by altering gene expression levels, which is the focus of the study by Luo and colleagues published in the June 2012 edition of the American Journal of Human Genetics. A second paper from Holt and colleagues published in the May 2012 edition of the European Journal of Human Genetics investigates an alternative mechanism whereby CNVs combine the 5' and 3' ends of two genes, creating a novel 'fusion gene'.

This pair of papers sheds light on some of the genomic imbalances that could contribute to ASD pathogenesis. Bioline offers products ideal for use in genome-wide studies of structural variants and disease susceptibility.

Luo and colleagues used genome-wide transcriptome profiling to evaluate the functional consequences of rare structural CNVs in ASD. The UCLA team along with collaborators from Yale, Wellcome Trust Sanger Institute, Carnegie Mellon, and University of Pittsburgh identified a number of interesting candidate ASD loci at 12p11.22, 15q23, 1p34.3, 3q27, and 3p26.2. For example, the 3p26.2 loci, found deleted, harbours three genes: ITPR1, SETMAR and SUMF1, all of which are down-regulated. Although none of these genes has been previously associated with autism, they are all functionally linked to the nervous system.

This study provides evidence that pathogenic structural variants have a functional impact via transcriptome alterations in ASDs at a genome-wide level. The authors also demonstrate the usefulness of integrating gene expression and mutation data to prioritize candidate genes disrupted by potentially deleterious alterations.

Fusion-proteins play an important role in cancer genetics, as exemplified by BCR-ABL in leukemia and TMPRSS2-ERG in prostate cancers. A team from the Wellcome Trust Centre for Human Genetics, University of Oxford addressed the question of how common CNV-induced fusion transcripts are and whether they may play a role in ASD susceptibility. Firstly, using the published Illumina 1 million SNP array data from the AGP consortium, they performed bioinformatic analysis of existing CNV calls. They then validated CNVs using qPCR.

No evidence was found that fusion-gene generating CNVs led to ASD susceptibility. However, the discovery of a MAPKAPK5-ACAD10 transcript with an estimated frequency of ~1/200 suggests that gain-of-function mechanisms should be considered in future studies of genomic imbalance and disease susceptibility.

The groundbreaking discovery by Sir Alexander Fleming of the first antibiotic penicillin in 1929, and its subsequent isolation by Ernst Chain and Sir Howard Florey in 1939, led to the award of the Nobel prize in 1945. This discovery revolutionized modern medicine, paving the way for the development of many more natural antibiotics such as chloramphenicol, streptomycin and tetracycline. These antibiotics were effective against the full array of bacterial pathogens including Gram-positive and Gram-negative bacteria, intracellular parasites and Mycobacterium tuberculosis.

However, over the past several decades, bacteria have developed resistance to existing drugs. Drug resistance by so-called "superbugs" is a severe public health problem, whilst development of new generation anti-microbials faces great challenges.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important nosocomial pathogens. For example, an estimated 89,785 invasive MRSA infections were associated with 15,249 deaths in 2008 in the United States. Even when the infection is successfully treated it can double the average length of a hospital stay and thereby increase healthcare costs. MRSA is able to produce a range of unique toxins causing severe infections such as endocarditis, osteomyelitis, pneumonia and septic shock in patients with open wounds, invasive devices and weakened immune systems.

One part of the problem is that bacteria and other microbial pathogens are very hardy and have developed different antimicrobial resistance mechanisms. Several interesting acronyms have been coined to describe the range of resistance from multi-drug resistant (MDR), extensively drug resistant (XDR), to totally drug resistant (TDR). The increasing use and misuse of existing antibiotics in human and veterinary medicine as well as agriculture has also exacerbated the problem. Antimicrobial resistance is genetically based; resistance is mediated by the acquisition of extrachromosomal genetic elements containing resistance genes, such as plasmids, transposable genetic elements and genomic islands, which are transferred between bacteria via horizontal gene transfer.

Last month, a new study published in the New England Journal of Medicine was able to identify which isolates of MRSA were part of a hospital outbreak using whole genome sequencing. Scientists from the Wellcome Trust Sanger Institute, University of Cambridge and Illumina sequenced the isolates within a timescale that could influence infection control and patient management. The authors created an artificial "resistome" of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing. They also created a “toxome” consisting of toxin genes.

Bioline offers a range products for use in microbial research and below we document some of the publications in which Bioline reagents were used to advance research.

The Petez-Osorio group used ImmoMix Red to describe a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. This MID-DRS assay reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as two days with reduced assay costs, preparation time and risks due to user errors.

The Rosato group at the Center for Molecular and Translational Human Infectious Diseases Research, Methodist Hospital Research Institute in Houston, USA conducts translational research focusing on multidrug-resistant bacterial pathogens. The aim of their study was to identify the molecular genetic causes of drug resistance and resistance gene spread in MRSA.

In this paper, Dr Rosato’s group used DNA microarrays and qRT-PCR to evaluate differential gene expression during HeR-HoR selection and found increased expression of the agr two-component regulatory system. Their findings reinforce the concept that increased expression of agr during HeR-HoR selection plays a critical role in regulating the ß-lactam-induced increased mutation rate in very heterogeneous MRSA strains.

In another exciting paper from Dr Rosato’s group at the Methodist Hospital Research Institute, Houston, they teamed up with researchers from Virginia Commonwealth University and Cubist Pharmaceuticals to investigate the molecular basis of resistance to daptomycin (DAP), a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed drug mechanisms include disruption of the bacterial membrane wall.

Differential gene expression analysis using SensiMix One-Step Kit also showed up of the two-component regulatory system vraSR. Crucially, this effect was related to the impact of vraSR and mprF mutations in the cell wall. Their work underscores the suggestion that alterations in these two genes contributes to DAP-resistance in this group of clinical MRSA strains.

The Morrissey group at the University of Leicester, UK conducts research into microbial genetics. One of their major areas of investigation is the Staphylococcus aureus adaptive response to antibacterial copper. Their results shows copper resistance varies considerably between clinical strains due to the carriage of an additional plasmid-encoded copper homeostasis mechanism, copBmco. Importantly, this plasmid has the potential to spread to other S. aureus strains. This is the first time that plasmid-encoded copper resistance has been reported and shown to be transferable between pathogenic bacteria isolated from humans.

The aim of this study at St. Vincent’s Hospital in Australia was to determine the utility of molecular methods compared to selective agars for MRSA detection. The conclusion of the van Hal group was that molecular detection methods for MRSA remain sensitive and rapid, but are associated with greater expense.

It's time for the second in our series of Bioline Scholar Monthly compilations. This month we're focusing on the many uses of Bioline polymerases in forensic science.

Identification of a report's species is one of the basic analyses in forensics. However, due to the nature of the sampling environment, DNA samples often contain PCR-inhibitory substances which may generate blank or incomplete DNA profiles. The common approach to overcoming PCR inhibition is extensive DNA purification, but this can increase the risk of DNA loss. In some cases, isolation of single cells using laser-capture microdissection can be used, but again this reduces the amount of DNA available.

DNA polymerases that can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition, without any additional sample preparation, are therefore advantageous, as are polymerases that result in high yields.

Bioline’s DNA polymerases are very robust and have been carefully designed to overcome these problems. BIO-X-ACT™ Short in particular is specifically designed for difficult/problematic PCR applications that require high processivity and fidelity, applications that would normally fail with other DNA polymerase. MangoTaq™ has also been designed for problematic and ancient DNA, whereas IMMOLASE™ and BIOTAQ™ are high yield for small sample sizes. Together with our new MyTaq™, MyFi™ and RANGER Bioline has polymerases to meet all the high-fidelity requirements of forensic science.

So, without further ado, here's...

Bioline Scholar Monthly: February 2012

Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In Barkway and colleagues procedure, BIO-X-ACT Short DNA Polymerase was initially used to successfully verify the LAMP primer pair for Eimeria species specificity using PCR.

Out of 19 polymerases, the best performance was exhibited by the Mango-Taq DNA polymerase, which was the only polymerase which was able to amplify the ~620 bp amplification product from the 102 year old sample.

In Australia and globally, Sarcophagidae flies remain unexploited as indicators of post-mortem interval in forensic investigations. A molecular identification method involving DNA ‘barcoding’ of the mitochondrial COI gene from 16 species of Australian Sarcophagidae was successfully developed. The authors conclude analysis of sarcophagids in forensic entomology should increase and their value as tools in criminal investigations realised.

Microsatellite markers were developed for the medicinal plant Tripterygium (Celastraceae) to assess its population structure and to facilitate source tracking of plant materials used for medicinal extracts.